Analytical Method Development and Validation of Ticagrelor from Bulk and Formulation
 
Anand Gupta*, Vaishali Jadhav, Ashish Jain
Shri. D. D. Vispute College of Pharmacy & Research Center, New Panvel

*Corresponding Author E-mail: anandgpt15@gmail.com

 

ABSTRACT:

Ticagrelor is a Platelet Aggregation Inhibitor which is white to off-white, highly crystalline and non-hygroscopic powder, having High Solubility and Low Permeability. A Simple, Rapid, Selective, Precise and Accurate UV and HPLC Method have been developed for the Estimation of Ticagrelor in bulk drugs and its Tablet dosage form. The max was found to be λmax 255 nm. As per the solution stability experiments, this drug was stable in the solvent composition of ACN:Methanol (85:15 v/v) and hence used as a diluent for sample preparation. The developed method was optimized to get reproducible results with minimum run time. The Stationary Phase was C18 (250 x 4.6 mm i.d., 5μ) Mobile Phase was ACN:Methanol (85:15 v/v), Flow rate 1.0 ml/min, Injection volume 10 µl, PDA detection at λmax 255nm and Run time 7 min. It provides a linear response over the Conc. Range of 5-25 µg/ml with Correlation Co-efficient of 0.999 and LOD and LOQ was found to be 0.20 μg/ml and 0.61 μg/ml respectively. %Recovery was found to be 99.06, 99.77, and 100.99% for the levels of 80, 100, and 120% respectively.  Both the Method was found to be Robust with better accuracy and Precision having % RSD value less than 2.

 

KEYWORDS: Ticagrelor; Validation; Method Development; Assay Method.

 

 


1. INTRODUCTION:

Ticagrelor ((1S,2S,3R,5S)-3-[7-[(1R,2S)-2-(3,4-Difluorophenyl)cyclopropylamino]-5-(propylthio)-3H-[1,2,3]triazolo[4,5-d]pyrimidin-3-yl]-5-(2-hydroxyethoxy)cyclopentane-1,2-diol) is Platelet Aggregation Inhibitor which Ticagrelor blocks adenosine diphosphate (ADP) receptors of subtype P2Y12. In contrast to the other antiplatelet drugs, ticagrelor has a binding site different from ADP, making it an allosteric antagonist, and the blockage is reversible.

 

 

 

Moreover, the drug does not need hepatic activation, which might work better for patients with genetic variants regarding the enzyme CYP2C19 (although it is not certain whether clopidogrel is significantly influenced by such variants). It absorbed quickly from the gut and the bioavailability is 36%. The drug is metabolized principally by Cytochrome P-450 (CYP) isoenzyme 3A4 to an active metabolite that has similar antiplatelet activity as the parent drug. Plasma concentrations of ticagrelor and its active metabolite increase in a dose-dependent manner with peak concentrations achieved within approximately 1.5 and 2.5 hours, respectively. The primary route of ticagrelor elimination is hepatic metabolism. When radiolabeled ticagrelor is administered, the mean recovery of radioactivity is approximately 84% (58% in faeces, 26% in urine). The most common side effects are shortness of breath (dyspnea, 14%) and various types of bleeding, such as hematoma, nosebleed, gastrointestinal, subcutaneous or dermal bleeding. Ticagrelor should be administered with caution or avoided in patients with advanced sinoauricular disease. They are decreased in patients that described themselves as ‘coloured’ and those with severe renal impairment. These differences are considered clinically irrelevant.

 

 

Fig 1: Chemical Structure of Ticagrelor

 

2. MATERIALS AND CHEMICALS:

2.1 Instrumentation:

Table No 1: Instruments Used

Sr No.

Name of instrument

Make and Model

1.

HPLC

Jasco Extrema LC System - 4000

2.

UV-Vis Spectrophotometer

Shimadzu 1800

3.

FTIR

Simadzu, IR Affinity – 1S, No. A219652

4.

Weighing Balance

Aczet (Sensitivity 0.0001 gm) Shimadzu (Sensitivity 0.001 gm)

5.

pH Meter

Lab India

6.

Sonicator

Life Care Equipment

7.

HPLC Column

C18 (250 x 4.6 mm i.d., 5µ)

8.

Digital pH Meter

Equiptronics EQ 610

 

2.2 Chemicals and reagents:

Table No 2: Chemicals Used

Sr No.

Name of Material

Manufacturer

Grade

1.

Acetonitrile

Research lab fine chem, Mumbai

HPLC

2.

Methanol

Research lab fine chem, Mumbai

HPLC

3.

Water

Research lab fine chem, Mumbai

HPLC

4.

o-phosphoric acid

Research lab fine chem, Mumbai

AR

5.

Disodium hydrogen phosphate

Research lab fine chem, Mumbai

AR

6.

Potassium dihydrogen phosphate

Research lab fine chem, Mumbai

AR

7.

Glacial acetic acid

Research lab fine chem, Mumbai

AR

8.

Membrane filter paper (0.45μ)

Pall Life Sciences, Mumbai

HPLC

9.

Syringe filter (0.45μ)

Research lab fine chem, Mumbai

HPLC

 

2.3   Preparation of Solutions:

2.3.1 Preparation of Standard Stock solution:

·      Weigh 10 mg of drug and transfer in 10 ml volumetric flask and add 5 ml of diluents in the flask. Shake well for 2 min and make up the volume up to the mark with diluent. This gives solution of 1000 µg/ml.

·      Pipette out 1 ml of above solution and transfer it to 10 ml volumetric flask and dilute it upto the mark with diluent to get concentration of 100 µg/ml

 

2.3.2 Preparation of Sample Stock solution:

·        Weigh and powder 10 tablets. Weigh a quantity of powder equivalent to 10 mg of Ticagrelor i.e. 34.54 mg of powdered drug and transfer in 10 ml volumetric flask and add 5 ml of diluent in the flask. Shake well and Sonicate for 10 min and make up the volume up to the mark with diluent. This gives solution of 1000 µg/ml.

·        Filter the above solution with suitable filter paper and pipette out 1 ml of solution and transfer it to 10 ml volumetric flask and dilute it upto the mark with diluent to get concentration of 100 µg/ml.

 

 


 


 

3. RESULTS AND DISCUSSIONS:

3.1 Drug Identification:

 

Fig 2: IR Spectra of Ticagrelor

 


 

 

The spectra of Ticagrelor shows the following groups at their frequencies and interpretation is given in Table 3,

 

Table 3: Interpretation of IR Spectra of Ticagrelor

Sr No.

Wavenumber (cm-1)

Interpretation

1.

1273.02

C=N

2.

1195.87

C-F

3.

1327.03

C-O (stretch) Ester

4.

1624.06

C=C (stretch) Aromatic

5.

1425.04

CH2 (bend)

6.

671.23

C-H (out-of-pane bend)

3.2 Selection of Detection Wavelength:

The standard solution of 10 µg/ml of Ticagrelor were prepared and scanned over the range of 400-200 nm. After scan was completed it shows highest absorbance and peak at wavelength of 255 nm. So, 255 nm was selected as the detection wavelength.

 

3.3 Assay Method:

Six replicates of Standard and Sample solutions were prepared and %Assay was calculated

 

 

Fig 3: UV Spectra of Ticagrelor

 

 

Fig 4: Chromatogram of Standard Ticagrelor

 

 

Fig 5: Chromatogram of Sample Ticagrelor

Observation Table:

Table 4: Assay Results of Ticagrelor by HPLC Method

Sr No.

Conc. (µg/ml)

Standard Area

Sample Area

1

10

337150

337150

2

10

335462

325632

3

10

332589

334598

4

10

345698

351420

5

10

332154

339856

6

10

332147

342152

Average

335866.66

338468

Tab. Avg Wt.

207.2 mg

Std. Wt.

10 mg

Sample Wt.

34.54 mg

Label Amount

60 mg

Std. Purity (%)

99.7

Assay (%)

98.91

 

Observation:

The amount of Ticagrelor present in taken dosage form was found to be 98.91%.

 

3.4 ChromatographicConditions:

a.      Column: C18 (250 x 4.6 mm i.d., 5μ)

b.      Wavelength: 255 nm

c.      Oven Temperature: 30oC

d.      Flow rate: 1.0 ml/min

e.      Injection Volume: 10 ml

 

3.5 Method Development and Validation:

3.5.1 Selection of Suitable Diluent:

The diluent was selected on the basis of its solubility studies performed on different solvents. So depending on Solubility study, Mobile Phase composition and Reproducible peaks diluent were selected. ACN: Methanol in the ratio of 85:15% v/v was selected as a diluent.

 

3.5.2 Selection of Mobile Phase:

A range of solvents were screened in an effort to get a well resolved, sharp and symmetrical peak for the drug. Hence, various numbers of trails were carried out to select a suitable mobile phase for the HPLC method development.

 

 

 

Fig 6: Chromatogram of Ticagrelor

 

3.5.3 Validation:

3.5.3.1 Specificity:

 

Fig 7: Blank Chromatogram for Specificity

 

 

Fig 8: Chromatogram of Ticagrelor for Specificity

 

Observation:

No interference of mobile phase, diluents, and impurities is observed and also no change in Retention time was observed and hence, the method was found to be Specific.

 

 

 

 

3.5.3.2 Linearity:

The linearity of Ticagrelor was found to be in the range of 5-25 µg/ml.

 

Table 5: Linearity Data of Ticagrelor by HPLC Method

Sr no.

Conc. (µg\ml)

Area

1.

0

0

2.

5

164266

3.

10

336735

4.

15

502576

5.

20

665644

6.

25

836735

Correlation Co-efficient

0.999

Y-Intercept

Y = 33449x-458.5

 

 

Observation:

The Correlation Co-efficient for linear curve obtained between Concentrations vs. Area for standard preparations of Ticagrelor was found to be 0.999. The relationship between the concentrations of Ticagrelor was linear in the range examined since all the points lie in a straight line and Correlation Co-efficient was well within limits.

 

 

Fig 9: Linearity graph of Ticagrelor by HPLC Method


Observation Table:

Table 6: Accuracy data of Ticagrelor by HPLC Method

Level

Stock Added

Standard Added

Volume Make up

Area

Conc. (µg/ml)

% Recovery

Mean

80%

10 ppm

8 ppm

10 ml

594509

17.75

98.66

99.06 ± 0.69

10 ppm

8 ppm

601695

17.97

99.85

10 ppm

8 ppm

594509

17.75

98.66

100%

10 ppm

10 ppm

672934

20.10

100.52

99.77 ± 0.73

10 ppm

10 ppm

667716

19.94

99.74

10 ppm

10 ppm

663096

19.81

99.05

120%

10 ppm

12 ppm

748302

22.35

101.62

100.99 ± 1.07

10 ppm

12 ppm

748173

22.35

101.60

10 ppm

12 ppm

734550

21.94

99.75

 

Table 7: Precision data of Ticagrelor by HPLC Method

Sr No.

Conc. (µg/ml)

System Precision

Method Precision

Area

1.

20

695315

695263

2.

20

690379

689956

3.

20

693872

701110

4.

20

692033

685623

5.

20

690080

695623

6.

20

691252

695485

Mean

692155.16

693843.33

Standard Deviation (SD)

2060.28

5354.76

Relative Standard Deviation (RSD)

0.0029

0.0077

%RSD

0.29

0.77

Table 8: Robustness data of Ticagrelor by HPLC Method

Sr no.

1.

2.

3.

Mean

SD

%RSD

Flow rate

0.8 ml/

min

Area

444323

445689

447855

445955

1781.03

0.39

Rt

3.87

3.85

3.85

3.85

0.01151

0.29

NTP

5586

5464

5562

5537

12.49

0.22

1.2 ml/

min

Area

292750

298569

289987

293768

1488.286

0.50

Rt

2.61

2.62

2.61

2.61

0.0057

0.22

NTP

4675

4658

4596

4646

41.581

0.89

Wavelength

252 nm

Area

353501

356985

356696

355727

1933.458

0.54

Rt

3.10

3.15

3.11

3.12

0.0264

0.84

NTP

5323

5333

5454

5370

16.921

0.31

258

Area

333335

335623

336562

335173

1659.829

0.49

Rt

3.10

3.11

3.15

3.12

0.0264

0.84

NTP

5323

5323

5322

5322

0.5773

0.01

Temperature

25oC

Area

349033

344585

348956

347524

2546.11

0.73

Rt

3.15

3.15

3.18

3.16

0.0173

0.54

NTP

5423

5444

5623

5496

16.70

0.30

35oC

Area

365142

365656

359865

363554

3205.376

0.88

Rt

3.05

3.08

3.05

3.06

0.0173

0.56

NTP

5164

5112

5123

5133

27.404

0.53

 

 

 


3.5.3.3 Accuracy (Recovery):

The recovery studies were carried out three times, Chromatogram was recorded and %Recovery and Mean %Recovery was calculated.

 

Observation:

%Recovery was found to be 99.06, 99.77, and 100.99% for the levels of 80, 100, and 120% respectively.

 

3.5.3.4 Precision:

Six replicates of Standard solution were prepared and %RSD was calculated.

 

Observation:

The %RSD for System and Method Precision was found to be 0.29 and 0.77% respectively.

 

3.5.3.5 Robustness:

The Three replicates of Standard Solution were prepared by varying the Wavelength (±3 nm), Flow rate (±0.2 ml/min), and Temperature (±5şC) and effect was checked for system suitability.

 

Observation:

The System Suitability was found to be within limit at different Wavelengths, Flow rates and Temperatures for Robustness study conducted.

 

3.5.3.6 LOD and LOQ:

LOD and LOQ were found to be 0.20 µg/ml and 0.61 µg/ml respectively.

 

3.5.3.7 System Suitability Test:

Injected single injection of blank and five replicate of Standard Solution. The %RSD of five replicate.

 

%RSD should not be more than 2.0%. The USP Tailing Factor for Ticagrelor peaks should not be more than 2.0. The USP Plates should not be less than 2000.

 

Table 9: Result of System Suitability Test

Sr. No

Parameter

Result

1.

Tailing Factor

1.03

2.

NTP

5789

3.

Retention time

3.12

4.

%RSD

0.77

 

Observation:

The system suitability was found to be within limits.

 

4. CONCLUSION:

Based on trial experiments conducted for Method Development, this drug was stable in the solvent composition of ACN: Methanol (85:15 v/v) and hence used as a diluent for sample preparation. The developed method was optimized to get reproducible results with minimum run time. The Stationary Phase was C18 (250 x 4.6 mm i.d., 5μ) Mobile Phase was ACN: Methanol (85:15 v/v), Flow rate 1.0 ml/min, Injection volume 10 ml, PDA detection at λmax 255nm and Run time 7 min. It provides a linear response over the Conc. Range of 5-25 mg/ml with Correlation Co-efficient of 0.999 and LOD and LOQ was found to be 0.20 μg/ml and 0.61 μg/ml respectively. %Recovery was found to be 99.06, 99.77, and 100.99% for the levels of 80, 100, and 120% respectively. Both the Method was found to be Robust with better accuracy and Precision having % RSD value less than 2. The proposed Method has been successfully used for the Routine analysis for the determination of assay in Ticagrelor Tablet.

 

5. REFERENCES:

1.     https://reference.medscape.com/drug/brilinta-ticagrelor-999674 (assessed on 1/10/2018)

2.     https://pubchem.ncbi.nlm.nih.gov/compound/Ticagrelor (assessed on 1/10/2018)

3.     http://www.thwink.org/sustain/glossary/AnalyticalMethod.html (accessed on 2/11/2018)

4.     https://www.quora.com/What-is-the-basic-principle-of-high-performance-liquid-chromatography-HPLC (assessed on 07/11/2018)

5.     http://www.chromatographyonline.com/mobile-phase-optimization-strategies-reversed-phase-hplc-0 (assessed on 14/11/2018)

6.     https://www.ich.org/products/guidelines/quality/article/quality-guidelines.html (assessed on 14/11/2018)

7.     Rajendra B. Kakde, et al. Method Development, Validation and Impurity Profiling Of Ticagrelor By Acid Degradation Method. IJPRIF. 2017, 10 (4), 225-236

8.     Md. ShabanaSulthana, et al. Development Of A Validated HPLC-PDA Method For Stability Indicating Study Of Ticagrelor: A Novel Anti-Platelet Agent (P2y12-Adp Receptor Blocker) IJPCBS. 2017, 7(1), 36-42

9.     Muddukrishna BS, et. al., Ticagrelor in bulk and comparison with published methods, Journal of Global Pharma Technology, 2016, 12(8), 01-06

10.   P R. Kulkarni, et al. Development and validation of RP-HPLC method for estimation of Ticagrelor in bulk form, IJRPC 2016, 6(4), 733-737

11.   Livia Maronesi Bueno, et al. HPLC method for simultaneous analysis of ticagrelor and its organic impurities and identification of two major photo degradant products, European Journal of Pharmaceutical Sciences 2017, 97, 22–29

12.   Vegesna Swetha, et al. Analytical method development and validation of stability indicating assay method of Ticagrelor tablets by using RP-HPLC, WJPMR, 2017, 10, 235-241.

 

 

 

 

 

Received on 14.03.2019        Accepted on 10.04.2019

© Asian Pharma Press All Right Reserved

Asian J. Pharm. Res. 2019; 9(3):141-146.

DOI: 10.5958/2231-5691.2019.00022.4